Peptides involved in the pathogenesis of HIV infection

ABSTRACT

This invention relates to peptides involved in the pathogenesis of human immunodeficiency virus (&#34;HIV&#34;). More particularly, this invention relates to peptides from the env region of the HIV genome and the use of such peptides in methods and compositions for preventing, treating, or detecting acquired immune deficiency syndrome (&#34;AIDS&#34;) infection.

This is a continuation of application Ser. No. 873,621, filed June 12,1986, now abandoned.

TECHNICAL FIELD OF INVENTION

This invention relates to peptides involved in the pathogenesis of humanimmunodeficiency virus ("HIV"). More particularly, this inventionrelates to peptides from the env region of the HIV genome and the use ofsuch peptides in methods and compositions for preventing, treating, ordetecting acquired immune deficiency syndrome ("AIDS") infection.

BACKGROUND ART

Acquired immune deficiency syndrome ("AIDS") is a disease characterizedby severe or, typically, complete immunosuppression and attendant hostsusceptibility to a wide range of opportunistic infections andmalignancies. AIDS' complete clinical manifestation is usually precededby AIDS related complex ("ARCS"), a syndrome accompanied by symptomssuch as lymphadenopathy, fever and weight loss.

The human immunodeficiency virus ("HIV") retrovirus is thought to be theetiological agent responsible for AIDS infection and the ARCS syndrome[M. G. Sarngadharan et al., "Detection, Isolation, and ContinuousProduction of Cytopathic Retroviruses (HTLV-III) From Patients With AIDSand Pre-AIDS", Science, 224, pp. 497-508 (1984)].* Between 85 and 100%of the AIDS/ARCS patient population test seropositive for HIV [G. N.Shaw et al., "Molecular Characterization of Human T-Cell Leukemia(Lymphotropic) Virus Type III In The Acquired Immune DeficiencySyndrome", Science, 226, pp. 1165-1170 (1984)].

Upon infection of a host, the primary targets of the HIV virus are T-4lymphocytes, also known as helper or inducer cells. T-4 lymphocytesinteract with other specialized cell types of the immune system toconfer immunity to or defense against infection. More specifically, T-4lymphocytes stimulate production of growth factors which are critical tothe functioning of the immune system. For example, they act to stimulateB cells, the descendants of hemopoietic stem cells, which promote theproduction of defensive antibodies. They also activate macrophages("killer cells") to attack infected or otherwise abnormal host cells,and induce monocytes ("scavenger cells") to encompass and destroyinvading microbes. Accordingly, when T-4 lymphocytes are renderednon-functional by HIV infection, this complex immune defense system isdestroyed and the host becomes susceptible to a wide range ofopportunistic infections. In addition to T-4 lymphocytes, the HIV virushas also been shown to infect central nervous system cells, macrophagesand B lymphocytes [J. M. Ismach, "AIDS: Can The Nation Cope", MedicalWorld News (Aug. 25, 1985)].

The genome of retroviruses such as HIV contains three regions encodingstructural proteins. The gag region encodes the core proteins of thevirion. The pol region encodes the virion RNA-dependent DNA polymerase(reverse transcriptase). The env region encodes the major glycoproteinfound in the membrane envelope of the virus and in the cytoplasmicmembrane of infected cells. The capacity of the virus to attach totarget cell receptors and to cause fusion of cell membranes are two HIVvirus properties controlled by the env gene. These properties arebelieved to play a fundamental role in the pathogenesis of the virus.

HIV env proteins arise from a precursor polypeptide that, in matureform, is cleaved into a large heavily glycosylated exterior membraneprotein of about 481 amino acids--gp120--and a smaller transmembraneprotein of about 345 amino acids which may be glycosylated--gp41 [L.Ratner et al., "Complete Nucleotide Sequence Of The Aids Virus,HTLV-III", Nature, 313, pp. 277-284 (1985).

In the absence to date of effective treatments for AIDS, many effortshave centered on prevention of the disease. Such preventative measuresinclude HIV antibody screening of all blood, organ and semen donors andeducation of AIDS high-risk groups regarding transmission of thedisease.

Experimental or early-stage clinical treatment of AIDS and ARCSconditions have included the administration of antiviral drugs, such asHPA-23, phosphonoformate, suramin and ansamycin, which apparentlyinterfere with replication of the virus by inhibiting its reversetranscriptase. Administration of some of these drugs in effectiveamounts has, however, been accompanied by undesirable and debilitatingside effects. Other proposed methods for treating AIDS have focused theadministration of alpha interferon or the application of hybridomatechnology. Most of these treatment strategies are expected to requirethe co-administration of immunomodulators, such as interleukin-2.However, while some of these treatments are promising, none has beenshown to be truly effective.

Recent studies have also demonstrated that HIV is experiencing geneticdrift in humans. At least two classes of the virus have now beenidentified in AIDS patients in the United States. Furthermore, patientshaving high levels of HIV neutralizing antibodies suffer more seriousforms of the disease than those patients with poor neutralizingcapabilities [Dr. William Haseltine, speech at Memorial Sloan-KetteringCancer Center, Oct. 9, 1985]. These recent observations suggest seriousobstacles to the development of an effective vaccine or monoclonalantibody-directed therapeutic method against HIV AIDS infections.

Accordingly, despite these developments to date, the need exists for thedevelopment of effective agents for the prevention, treatment anddiagnosis of HIV and AIDS-related infections.

DISCLOSURE OF THE INVENTION

The present invention solves the problems referred to above by providingpeptides involved in the pathogenesis of the HIV virus. According to oneembodiment, the peptides of this invention are selected from the groupconsisting of peptides characterized by an amino acid sequence derivedsubstantially from the region between about amino acid 600 and aminoacid 750 of the HIV env gene. This region is believed to have animportant role in virus-mediated pathogenic events. More preferably, thepeptides of this invention consist substantially of the following aminoacid sequences of the HIV env gene--peptide 1: amino acids 616-632;(PWNASWSNKSLEQIWNN); peptide 2: amino acids 667-680; (LLELDKWASLWNWF);peptide 3: amino acids 702-715 (LRIVFAVLSVVNRV) and peptide 4: aminoacids 728-751 (LPIPRGPDRPEGIEEEGGERDRDR). Another embodiment of thisinvention includes peptide 5, which consists substantially of aminoacids 426-450 (RIKQIINMWQEVGKAMYAPPISGQI) of the HIV env gene. Thesepeptides produce antisera which, in conventional assays, bind to the HIVvirus, inhibit syncytium formation or neutralize the virus. In addition,the peptides themselves may be capable of inhibiting HIV-directedsyncytium formation or neutralizing HIV in conventional assays. Suchpeptides, therefore, are useful in compositions and methods forpreventing, treating and detecting AIDS infection.

The peptides of this invention comprise functional regions of the HIVenv protein involved in virus-mediated events, such as adsorption tonormal cells and syncytium formation, which contribute to thepathogenesis of the disease. The functional regions encompassed by thesepeptides also correspond to immunogenic determinants of the HIV env genewhich are highly conserved. Accordingly, these peptides comprisesegments of HIV env protein which are highly immunogenic and areinvolved in virus pathogenesis over the range of genetic variants of theHIV virus.

The peptides of this invention may be advantageously used in vaccines ortherapeutic compositions which elicit antibodies reactive with thenative env protein of the HIV virus or which interfere with the virus byneutralization or inhibition of syncytium formation. Furthermore, thesepeptides are easily modified in composition and conformation to improvethe specific activity of those peptides against the HIV virus. Inaddition, these peptides may be used as diagnostic agents for detectingHIV infections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequences of each of peptides 1-5 of thisinvention, as well as that of the region between amino acid 600 andamino acid 750 of the HIV env gene. In this figure, the amino acids arerepresented by single letter codes as follows:

    ______________________________________                                        Phe: F      Leu: L     Ile: I     Met: M                                      Val: V      Ser: S     Pro: P     Thr: T                                      Ala: A      Tyr: Y     His: H     Gln: Q                                      Asn: N      Lys: K     Asp: D     Glu: E                                      Cys: C      Trp: W     Arg: R     Gly: G                                      ______________________________________                                    

DETAILED DESCRIPTION OF THE INVENTION

Referring now to FIG. 1, we prepared various peptides corresponding tosegments of the env gene of the HIV genome and tested them in severalconventional assays to demonstrate that they display activitiesreflecting their involvement in virus-mediated pathogenic events.

It should be understood that the present invention is not limited to theillustrative peptides depicted in FIG. 1. Instead, a peptide fallingwithin the scope of this invention may extend outside of or compriseless than the region between amino acid 600 and amino acid 750 of theHIV env gene, as long as a substantial part of that peptide ischaracterized by an amino acid sequence from that region, or segments orcombinations thereof, and that peptide demonstrates the desiredimmunological or biological activity against HIV. In addition, peptidesaccording to this invention include those having amino acid sequenceswhich are longer or shorter in length than those of peptides 1-4 orwhich comprise segments or combinations thereof, as long as suchpeptides consist substantially of the region between amino acids 600-750of the HIV env gene and demonstrate the desired immunological orbiological activity. Furthermore, peptides according to this inventioninclude those characterized by a sequence of amino acids which is longeror shorter than that of peptide 5, or which comprise segments of thatpeptide and which display immunological or biological activity againstHIV.

Accordingly, it should be understood that the specific selection of anyone peptide within the peptides of this invention is not critical. Sucha selection may be carried out by taking a number of peptides andtesting them for their immunological and biological activity against HIVas described herein.

The peptides according to this invention may be prepared by conventionalsynthesis using any of the known peptide synthesis methods, includingsynthesis on a solid support. The peptides of the invention may also beprepared in appropriate hosts transformed with DNA sequences that codefor the desired peptide. For example, a peptide of this invention may beprepared by the fermentation of appropriate hosts that have beentransformed with and which express a DNA sequence encoding that peptide.Alternatively, DNA sequences coding for several of the peptides of thisinvention may be linked together and those sequences may then be used totransform appropriate hosts to permit the expression of peptidesinvolved in the pathogenesis of HIV infection. A combination of suchmethods may also be employed. In a preferred embodiment of thisinvention, chemical synthesis alone is employed. By means of thatmethod, the peptides of this invention are additionally advantagedbecause they are easily purified and are non-biological in origin.

Although some of the peptides of this invention, such as peptides 2 and3, may be employed alone in the compositions and methods describedherein, others are preferably coupled to one or more carrier proteins,such as keyhole limpet hemocyanin ("KLH") before use. The peptides arecoupled to the carrier protein in various conventional ways, such asthose described by M. Reichlin, "Use Of Glutaraldehyde As A CouplingAgent For Proteins And Peptides", Methods In Enzymology, 70, pp. 159-65(1980).

After preparing the peptide and coupling it to the carrier protein, ifdesired, the antigen is employed in the methods and compositions of thisinvention in a conventional manner. For example, the peptide or coupledpeptide, alone or in combination with other peptides of this invention,is usually mixed with one or a combination of well-recognized adjuvantsand additives, preferably by first dissolving the peptide, for example,in PBS with 0.1% SDS. In other embodiments of this invention, thepeptides may be linked to hydrophobic groups to build the adjuvant intothe composition. Of course, it should be understood that otherwell-known methods of preparing therapeutic compositions may be employedusing the peptides of this invention.

The above-prepared compositions are then employed in a conventionalmanner for the treatment of HIV infections. Such methods of treatmentand their dosage levels and requirements are well-recognized in the artand may be chosen by those of skill in the art from available methodsand techniques. For example, the peptides of this invention may becombined with a pharmaceutically acceptable adjuvant for administrationto an HIV-infected patient in a pharmaceutically acceptable manner andin an amount effective to lessen the severity of the HIV infection. Thedosage and treatment regimens will depend upon factors such as thepatient's health status, the severity and course of infection and thejudgment of the treating physician.

Alternatively, the peptides of this invention are useful in vaccines andmethods for protecting humans against HIV infection for at least someperiod of time. The peptides may be employed in these vaccines andmethods either alone or together with other peptides of this inventionin a manner consistent with the conventional utilization of antigens invaccines. For example, the peptides of this invention may be combinedwith pharmaceutically acceptable adjuvants conventionally employed invaccines and administered in immunologically effective amounts toprotect patients for some time against HIV infection.

Both the compositions and vaccines of this invention may be administeredto patients via conventional modes of administration. The frequency ofadministration will depend upon factors such as the particularcomposition or vaccine employed and the condition of the patient. Theneed for subsequent treatments with these compositions or boosters ofthese vaccines will depend upon the results of the initial treatment orvaccination.

In addition, the peptides of this invention and the antibodies raised tothem may be employed in presently available methods and kits designed todetect the presence of HIV and antibodies to HIV in blood, organ orsemen samples.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLE I A. Preparation Of Peptides Involved In The Pathogenesis of HIVInfection

We synthesized peptides 1-5, corresponding to segments of the env geneof the HIV genome. These peptides are depicted in FIG. 1, in which theamino acid numbering corresponds to that set forth for the env gene inL. Ratner et al., "Complete Nucleotide Sequence Of The AIDS Virus,HTLV-III", Nature, 313, pp. 277-284 (1985).

We synthesized the peptides using an improved version of the solid phasemethod described by R. B. Merrifield, "Solid Phase Peptide Synthesis. I.The Synthesis Of A Tetrapeptide", J. Am. Chem. Soc., 83, pp. 2149-54(1963), using an Applied Biosystems Model 430A peptide synthesizer andreagents and procedures as supplied by producer. In this improvedmethod, we deblocked and cleaved the protected peptides from the resinwith liquid HF containing 10% anisole, in a variation of the methoddescribed by S. Sakakibara et al., "Use Of Anhydrous Hydrogen FluorideIn Peptide Synthesis. I. Behavior of Various Protective Groups InAnhydrous Hydrogen Fluoride", Bull. Chem. Soc. Jap., 40, pp. 2164-68(1967).

We first purified the peptides cleaved from the resin by partitionchromatography on a Sephadex LH20 column using n-Butanol/water (6/100)as eluent. The eluate was further purified by semipreparative highpressure reversed phase chromatography on an Altex Ultrasphere-ODScolumn, by elution with a 0.1% TFA acetonitril gradient. After wehydrolyzed the eluate with 6N HCl for 18 hours, we carried out aminoacid analysis on a Beckman amino acid analyzer to confirm the amino acidsequences of the peptides produced.

B. Immunological Activity Of The Peptides Of This Invention

1. Coupling Of Peptides To Carrier Proteins

In one embodiment of this invention, peptides 1 and 4-5 were preferablycoupled to one or more carrier proteins before use. Accordingly, wecoupled each of those peptides prepared as described above to thecarrier protein keyhole limpet haemocyanin (KLH, Sigma) by mixing 2 mgof peptide in 2 ml sodium phosphate buffer (0.1M, pH=8) with 5 mg of KLHin 2 ml sodium phosphate buffer (0.1M, pH=8). We then added 1 ml of a0.25% glutaraldehyde solution to the mixture in several portions over aperiod of 1 hour. We stirred the resulting mixture for another 6 hoursand then dialyzed it against PBS overnight.

For use in vaccine compositions, the peptides of this invention may becoupled with tetanus toxoid antigen, diphteria toxoid antigen or anothernatural or synthetic carrier suitable for use in humans usingconventional techniques. Alternatively, the peptides may be coupled witha suitable adjuvant to enhance the immune response in the patient. Thepeptides may also be used in combination with any suitable synthetic lowmolecular weight carrier before use. Finally, an additional cysteineresidue may be added to the C or N terminus of the peptide for couplingto a suitable carrier by disulfide linkage.

2. Inoculation Of Test Animals

We emulsified each of the KLH-coupled peptides 1, 4-5 and non-coupledpeptides 2 and 3 with Freund's complete adjuvant a 1:1 ratio.Subsequently, we inoculated groups of 3 BALB/CJ mice (JacksonLaboratory, Bar Harbor, Me.) by subcutaneous injection of 100 μg/250 μlof the emulsification into each mouse. On following days 14 and 35, eachmouse received a booster injection of 100 μg/250 μl of the same coupledpeptide emulsified 1:1 in Freund's incomplete adjuvant. Tail bleeds weretaken on days 21 and 42, with serum samples being stored at -20° C.until the time of assay.

3. Immunological Assays

i. ELISA With Antipeptide Sera Against env Peptide Coated Plates

In this assay, we determined that antiserum raised in an animal by eachof peptides 1-5 of this invention binds to that peptide. Accordingly,the peptides of this invention are immunogenic and elicit a response intest animals.

To carry out the assay, we coated two of four 96-well microtiter plates(Nunc Immuno I) with 50 μl of a mixture of 50 μg/ml uncoupled peptide inPBS (20 mM phosphate, 150 mM NaCl, pH=7.2) and incubated the plates overnight at room temperature. The third and fourth plates, which served ascontrols for, respectively, the first and second plates, were treatedidentically to those plates but were not pre-coated with peptide. Wethen inverted the plates to empty all wells and washed the plates 3times with PBS/0.05% Tween-20. The plates were blotted dry by gentletapping over paper towels. After blotting the plates, we added 350 μl ofa 5% fetal calf serum/PBS ("FCS/PBS") solution to each well andincubated the plates for 1 hour at room temperature. We then washed andblotted the plates as before.

We then assayed serum samples pooled from each group of 3 mice on thetwo pre-coated plates prepared as described above and on two controlplates. In the first pre-coated plate, we assayed the antibody responseto the immunogen peptide at an initial dilution of 1:10, followed byserial 2-fold dilutions in 5% FCS/PBS. In the second plate, an initialserum dilution of 1:20 was followed by serial 3-fold dilutions in 5%FCS/PBS.

After a 2 hour incubation period, we washed the plates and blotted themdry as described above. We then added 50 μl of 1:2000 dilution of goatantimouse-IgG horseradish peroxidase ("HRP") (A.P., heavy and lightchain specific, Cappel Laboratories) in 5% FCS/PBS to each well andincubated the plates at room temperature for 1 hour. We then washed theplates with PBS/0.05% Tween-20. We added 42 mM of3,3',5,5'-tetramethylbenzidine in dimethylsulfoxide ("TMB/DMSO"), 7.35μl of 30% hydrogen peroxide ("H₂ O₂ ") to 50 ml of 0.1M sodiumacetate-citric acid buffer (pH=4.92). Subsequently, we added 50 μl ofthis solution to the wells using a 12 channel multiple pipet. We stoppedthe enzyme reactions with 50 μl of 2M H₂ SO₄ when the less dilutesamples reached an absorbance of 0.2 O.D. at 650 nm. We then analyzedthe plates spectrophotometrically at 410 nm using a microplate reader(Dynatech MR600) and observed that antiserum against each of peptides1-5 binds to that peptide.

ii. ELISA With Antipeptide Sera Against Virus-Coated Plates

In this assay, we demonstrated that antisera raised against the peptidesof this invention binds to HIV virus-coated plates.

We coated 96 well microtiter plates (Nunc Immuno I) with 100 μl of amixture of 5 μg/ml authentic HIV virus in carbonate buffer (pH=9.6) andincubated the plates overnight at 4° C. We then inverted the plates toempty all wells and washed the plates 3 times with deionized water.Virus-coated microtiter plates are also available fromElectronucleonics, Fairfield, N.J.

We blotted the plates again, and added 100 μl of carbonate buffer(pH=9.6) containing 5% bovine serum albumin to each well and incubatedthe plates at room temperature. Subsequently, we rinsed the plates 3times with deionized water.

After blotting the plates, we added 100 μl of saline-PO₄ (PBS)containing 20% normal goat serum to each well. We next added 5 μl ofhuman test serum or control serum to each well and incubated the platesovernight at 4° C., or for 2 hours at room temperature. We then washedthe plates 3 times with PBS containing 0.05% Tween-20 and blotted them.We next added 100 μl of a 1:4000 dilution of 1% normal goat serum andgoat anti-human-IgG HRP (heavy and light chain specific) in 0.05%PBS-Tween 20 to each well and incubated the plates for 1 hour at roomtemperature. We had titrated the anti-human-IgG HRP before use to assurea proper final concentration of indicator antibody. At the end of thehour incubation period, we rinsed the plates 2 times with PBS-Tween-20and once with plain PBS. We then added 100 pl of a solution of 0.005%and 0.05% orthophenylene diamine ("OPD")* in Sorenson's phosphatecitrate buffer (pH=5) and allowed reaction for minutes at roomtemperature in the dark.

We stopped the reaction by adding 50 μl of 4N H₂ SO₄ to each well. Theplates were read by visual inspection or using a microplate reader at490 nm.

Each plate had a series of "blank" control wells containing no humanserum or anti-human IgG-HRP conjugate and to which one of the followinghad been added:

saline-PO₄ (PBS) containing 20% normal goat serum

PBS-Tween-20 (0.05%)

Sorenson's phosphate-citrate buffer (pH=5) containing 0.005% H₂ O₂ and0.05% OPD.

In addition, each plate had a series of "background" control wellscontaining no human serum and to which one of the following had beenadded:

saline-PO₄ (PBS) containing 20% normal goat serum

PBS-Tween-20 (0.05%) containing 1% normal goat serum and goat -anti-human-IgG HRP at a dilution of 1:4000

Sorenson's phosphate-citrate buffer (pH=5) containing 0.005% H₂ O₂ and0.05% OPD.

Each test plate also had a negative and positive control serum.

Analysis of the plates revealed that antiserum raised against each ofpeptides 1, 2 and 4 of this invention binds to the HIV virus. Althoughantisera against peptides 3 and 5 did not bind to virus coated plates inthis assay, we believe that upon repetition of the assay, these antiserawould exhibit the binding activity demonstrated by peptides 1, 2 and 4.

4. Virus Functional Assays

i. Syncytium Inhibition Assay

We assayed the peptides of this invention, as well as antisera raised tothem, for their ability to inhibit syncytium formation in a variation ofthe assay procedure set forth in C. D. Richardson and P. W. Choppin,"Oligopeptides That Specifically Inhibit Membrane Fusion ByParamyxoviruses: Studies On The Site Of Action", Virology, 131, pp.518-32 (1983). In our assay, we added recombinant HIV env protein,instead of live virus, to cultures of T-4 positive cells in the presenceof one of the peptides of this invention, or antiserum raised thereto,and observed the degree of inhibition of syncytium formation and cellfusion in the cultures.

We demonstrated by this assay that the peptides of this invention, aswell as antisera raised to them, inhibit virus-mediated events, such asvirus adsorption to cells and syncytium formation. In the assay, peptide3, the only peptide tested, inhibited syncytium formation. We believethat peptides 1, 2, 4 and 5 would demonstrate the same activity in thisassay. Additionally, antiserum raised against each of peptides 1, 2, 4and 5 inhibited syncytium formation. Upon repetition of this assay, webelieve that antiserum against peptide 3 would also exhibit suchactivity.

Accordingly, this assay indicates the utility of the peptides of thisinvention and the antisera raised thereto as therapeutic agents. Forexample, administration of such peptides to an infected host may inhibitcell-to-cell transmission of the virus and virus-induced cell fusionsufficiently to prevent spread of the infection and ultimate destructionof the immune system. Alternatively, these peptides may be usefullyadministered in a priming dose which would permit a subsequentlyinfected host to raise neutralizing antibodies effective against thevirus.

ii. Virus Neutralization Assay

a. HIV Neutralization Based On Lysis Of Cells

We assayed antisera raised to the peptides of this invention, todetermine their ability to neutralize HIV virus based on lysis of cells.In this assay, we mixed HIV-sensitive cells with the antisera, incubatedthem for several days and then observed the cells microscopically forlysis.

We observed that antisera to peptides 1, 2 and 5 of this inventionneutralized HIV virus, preventing HIV infection and subsequent lysis ofcells. Although antiserum to peptide 4 did not display such activity andantiserum to peptide 3 was not tested, we believe that these antiserawould exhibit neutralization activity in further assays. Furthermore,although we did not assay the peptides of this invention, we believethat they would demonstrate neutralization activity. Such neutralizingactivities indicate that the peptides of this invention and antiserathereto are useful in vaccines for preventing HIV infection.Alternatively, these peptides and antisera are useful in therapeuticcompositions for inhibiting virus replication in an infected host.

C. Use Of The Peptides Of This Invention And Their Antibodies InDetecting HIV And Antibodies To HIV

Methods and diagnostic kits are presently available which are designedto detect the presence of HIV and antibodies to HIV. Peptides involvedin the pathogenicity of HIV infection prepared by the processes of thisinvention and antibodies raised with them can also be employed in thesemethods and kits to detect the presence of HIV and antibodies to HIV.These peptides and their antibodies may be packaged in diagnostic kitswhich allow the rapid and convenient identification of AIDS carriers.

For example, the peptides of this invention or antibodies raised usingthem can be employed in the immunological diagnostic tests currentlyavailable for HIV antigen or antibody detection, e.g., radio-immunoassayor ELISA techniques.

In each assay, both the peptides of this invention and antibodies tothese peptides, are used. The antibodies are produced by injectinglaboratory animals with the peptides of this invention in a suitablesolution, such as Freund's adjuvant, followed by bleeding the animalsome six weeks later, removing the red blood cells by centrifugation,and using the resulting serum. Alternatively, monoclonal antibodies tothe peptides of this invention may be produced using standard hybridomatechniques.

In one type of radioimmunoassay, antibodies to an HIV peptide producedas above are attached to a solid phase, for example, the inside of atest tube. A sample of the patient's serum is added to the tube,together with a known amount of a peptide of this invention, produced asabove, and labelled with a radioactive isotope such as radioactiveiodine. Any HIV antigen in the patient's serum will compete with thelabelled peptide for binding with the HIV antibodies. The excess liquidis removed, the test tube washed, and the amount of radioactivitymeasured. A positive result; i.e., that the patient's serum contains HIVantigen, is indicated by a low radio-activity count left in the tube, ascompared with a control.

In one type of ELISA test, a microtiter plate is coated with a peptideprepared in accordance with this invention, and to this is added asample of patient's serum. After a period of incubation permittinginteraction of any HIV antibody present in the serum with the HIVantigen, the plate is washed. A preparation of anti-human antibodies,raised in a laboratory animal by injection of semi-purified humanimmunoglobulin, and then linked to an enzyme, is added. Incubationallows an antibody-antigen reaction to take place, and the plate is thenrewashed. Thereafter, enzyme substrate is added to the microtiter plateand incubated for a period of time to allow the enzyme to react with thesubstrate. The absorbance of the final preparation is then measured. Alarge change in absorbance indicates a positive result, i.e., that thepatient's serum contains antibodies to HIV.

While we have hereinbefore presented a number of embodiments of thisinvention, it is apparent that our basic construction can be altered toprovide other enbodiments which utilize the process of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the claims appended hereto rather than the specificembodiments which have been presented hereinbefore by way of example.

tic kits are presently available which are designed to detect thepresence of HIV and antibodies to HIV. Peptides involved in thepathogenicity of HIV infection prepared by the processes of thisinvention and antibodies raised with them can also be employed in thesemethods and kits to detect the presence of HIV and antibodies to HIV.These peptides and their antibodies may be packaged in diagnostic kitswhich allow the rapid and convenient identification of AIDS carriers.

For example, the peptides of this invention or antibodies raised usingthem can be employed in the immunological diagnostic tests currentlyavailable for HIV antigen or antibody detection, e.g., radio-immunoassayor ELISA techniques.

In each assay, both the peptides of this invention and antibodies tothese peptides, are used. The antibodies are produced by injectinglaboratory animals with the peptides of this invention in a suitablesolution, such as Freund's adjuvant, followed by bleeding the animalsome six weeks later, removing the red blood cells by centrifugation,and using the resulting serum. Alternatively, monoclonal antibodies tothe peptides of this invention may be produced using standard hybridomatechniques.

In one type of radioimmunoassay, antibodies to an HIV peptide producedas above are attached to a solid phase, for example, the inside of atest tube. A sample of the patient's serum is added to the tube,together with a known amount of a peptide of this invention, produced asabove, and labelled with a radioactive isotope such as radioactiveiodine. Any HIV antigen in the patient's serum will compete with thelabelled peptide for binding with the HIV antibodies. The excess liquidis removed, the test tube washed, and the amount of radioactivitymeasured. A positive result; i.e., that the patient's serum contains HIVantigen, is indicated by a low radio-activity count left in the tube, ascompared with a control.

In one type of ELISA test, a microtiter plate is coated with a peptideprepared in accordance with this invention, and to this is added asample of patient's serum. After a period of incubation permittinginteraction of any HIV antibody present in the serum with the HIVantigen, the plate is washed. A preparation of anti-human antibodies,raised in a laboratory animal by injection of semi-purified humanimmunoglobulin, and then linked to an enzyme, is added. Incubationallows an antibody-antigen reaction to take place, and the plate is thenrewashed. Thereafter, enzyme substrate is added to the microtiter plateand incubated for a period of time to allow the enzyme to react with thesubstrate. The absorbance of the final preparation is then measured. Alarge change in absorbance indicates a positive result, i.e., that thepatient's serum contains antibodies to HIV.

While we have hereinbefore presented a number of embodiments of thisinvention, it is apparent that our basic construction can be altered toprovide other enbodiments which utilize the process of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the claims appended hereto rather than the specificembodiments which have been presented hereinbefore by way of example.

We claim:
 1. A peptide involved in the pathogenesis of the HIV virusselected from the group consisting of peptides consisting of amino acidsequences of the formulae: PWNASWSNKSLEQIWNN and LLELDKWASLWNWF.
 2. Apeptide involved in the pathogenesis of the HIV virus characterized by asequence of amino acids consisting of the formula:

    RIKQIINMWQEVGKAMYAPPISGQI.


3. A peptide involved in the pathogenesis of the HIV virus characterizedby a sequence of amino acids consisting of the formula:

    LPIPRGPDRPEGIEEEGGERDRDR.